Mir-142-3P regulates MAPK protein family by inhibiting 14-3-3η to enhance bone marrow mesenchymal stem cells osteogenesis

Clinical studies have found 14-3-3η to be associated with osteoporosis through undefined mechanisms. We aimed to investigate the role of 14-3-3η in osteoporosis and its potential associations with miRNAs. The Gene Expression Omnibus(GEO) and Human Protein Atlas 1 databases were analyzed to examine both the mRNA and protein expression of 14-3-3η in OP. Gene enrichment analyses were performed to explore the underlying mechanism of 14-3-3η based on DAVID. miRWalk was used to predict the associated miRNAs. The statistics were analysed by R software and SPSS software. 14-3-3η was overexpressed and knock down expressed in BMSCs by lentiviral vector transfecting. And BMSCs were induced by hypoxia. qRT-PCR and Western-Blot verified the expression of mRNA and protein. Scratch assay detected the migration of osteocytes. Co-immunoprecipitation and luciferase assay studied the 14-3-3η targeted protein and miRNA. overexpression and knock down of miRNA to verify the relationship of 14-3-3η and target genes. The 14-3-3η mRNA expression level was low in patients with osteoporosis, as corroborated by immunohistochemical staining images. Functional analyses revealed enrichment of the MAPK-associated cascade. 14-3-3η was correlated with MAPK family proteins and five key miRNAs, including mir-142-3p. In addition, 14-3-3η knockdown in BMSCs increased the mRNA and protein expression levels of Hif-α, VEGF, BMP-2, OPN, OST, and Runx2, and enhanced the cells migration ability. Under hypoxic conditions, Hif-α and BMP-2 protein expression levels were upregulated, whereas those of 14-3-3η and MAPK3 were downregulated. Co-immunoprecipitation experiments showed decreased binding of 14-3-3η to MAPK3. 14-3-3η knockdown produced the same results as hypoxia induction. Adding caspase3 inhibitor and knocking down 14-3-3η again prevented MAPK3 cleavage by caspase3 and inhibited BMP-2 expression. Moreover, under hypoxic conditions, miR-142-3P expression was upregulated and luciferase assays revealed 14-3-3η as its target gene. miR-142-3P overexpression decreased mRNA and protein levels of 14-3-3η and MAPK3, while increasing BMP-2 expression. miR-142-3P knockdown reversed these results. BMSC osteogenesis was suppressed by 14-3-3η, whereas miRNA-142-3p promoted it through the inhibition of 14-3-3η.

Target miRNA prediction 14-3-3η-related miRNAs were identified by using miRWalk 17 , a web interface with precise tools for predicting miRNA-target interactions.

Cell cultures
BMSCs were prepared by the Cellcook Company (Guangzhou, Guangdong, China).They were derived from large male dog (12-16 kg, 6-8 weeks old) iliac marrows provided by Laboratory Animal Center of Anhui Medical University.Bone marrow puncture was performed at the iliac crest and 5 mL of marrow were extracted and mixed with 5 mL of Dulbecco's modified Eagle media (DMEM).The mixture was centrifuged at 1000 r/ min for 8 min and the precipitate was retained.The cells were resuspended in 15 ml DMEM supplemented with 10% (v/v) fetal bovine serum (FBS; Biological Industries, Israel), 1% (w/v) penicillin (Invitrogen, Carlsbad, CA, USA), and 1% (w/v) sodium pyruvate (Invitrogen), and then transferred into a cell culture flask.The cells were incubated at 37 °C with 5% CO 2 for 48 h until adherent growth.Once the cells reached 90% confluence, they were digested with 0.25% (v/v) trypsin and subcultured serially.BMSCs grew adhering to the flask wall and displayed a fusiform or polypseudopod fibroblast morphology.The analysis of identification was performed with a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) using CD29 and CD34 as specific stem cell markers (BMSCs are CD29-positive and CD34-negative).
Our Ethics Committee approved this study.Written informed consents were obtained from all data donors.

Hypoxia induction in BMSCs
We placed the cell culture plates in an airtight container and exposed the cells to hypoxic conditions at specific timepoints.A mixed gas of 95% N 2 and 5% CO 2 was charged through the container for 30 min and sealed the air inlet/outlet.BMSCs were placed into separate hypoxia boxes during the intervention.

Lentivirus transductions
Sh14-3-3η lentiviral particles or control lentiviral particles were prepared by the Sigma Company (St. Louis, MO, USA).For lentiviral infections, we incubated BMSCs with lentiviral particles with 8 μg/mL of polybrene for 24 h.Following the incubation, we replaced the infection medium with fresh culture medium.

RNA extraction and qRT-PCR
The total RNA were isolated from cells using TRIzol™ reagent (Invitrogen).After reverse transcription into cDNA with a reverse transcription kit (Takara, Kusatsu, Shiga, Japan), we set up qRT-PCR reactions using TB Green®Premix Ex Taq™ (Takara) according to the manufacturer's instructions.The 14-3-3η gene-specific primers and miR-142-3P were synthesized by Sangon (Shanghai, China).qRT-PCR cycling conditions included an initial denaturing step (94 °C for 5 min), a denaturation extension (30 cycles at 94 °C for 40 s, followed by 57 °C for 50 s), an annealing step (72 °C for 1 min), and an extension step (72 °C for 5 min).We used the expression of beta-actin as an internal reference to normalize expressions of 14-3-3η.We carried out the qRT-PCR reactions in an ABI Step One Plus instrument (Applied Biosystems, Foster City, CA, USA).All the primer sequences are listed in the Supplementary Table 1 section.

Co-Immunoprecipitation (Co-IP) experiments
We added 500 µL PBS and protein A/G beads to 1.5 mL transferring tubes.We mixed the tubes with 14-3-3η, MAPK3, or control antibody, and placed them on a low-speed rotating shaker for 2-3 h at room temperature.Next, we centrifuged the tubes at 14,000 g 4 °C for 15 min and transferred the supernatants to the new tubes.We then washed the protein A/G-agarose beads twice with PBS and prepared a 50% protein A/G agarose working solution (in PBS).We added 100 μL of the solution to each 1-mL sample and shook them on a horizontal shaker for 10 min.We centrifuged the tubes at 14,000 g 4 °C for 15 min and transferred the supernatants to new tubes.The protein A/G agarose beads were washed 2-3 times.We quantified the total protein in each sample and diluted it to 1 μg/μL with PBS to decrease the detergent concentration.We then added buffer and lysed cells at 4℃ for 1 h and centrifuged the samples at 14,000 rpm for 15 min, before mixing the supernatants with protein A/G beads coupled with 14-3-3η, MAPK3, or control antibody.We added appropriate amounts of primary antibody and incubated the antigen-antibody complex samples on a rotating shaker at 4 °C overnight.The next day, we centrifuged the samples at 14,000 g for 3-5 s, collected the pellets, and washed them with pre-chilled washing buffer three times.We collected the supernatants and heated them at 95 °C for 8 min before proceeding to SDS-PAGE.

Dual-luciferase reporter assay
We designed and amplified the 14-3-3η3′-UTR sequence in Plko.1 plasmids by PCR and sub-cloned it into the psiCheck2.0vector for luciferase reporter assays and named the resulting constructs psi-14-3-3η3′-UTR-WT and psi-14-3-3η3′-UTR-Mut.Briefly, we incubated 293 T cells in 24-well plates and co-transfected them with 100 ng of psi-check 2.0 luciferase vectors containing the wild-type or mutated 14-3-3η3′-UTR, and miR-142-3P mimics/inhibitor or normal control according to the experimental groups.We performed dual-luciferase reporter assays (Promega, Madison, WI) according to the manufacturer's instructions.We performed the transfection experiments in triplicate.

Wound healing assays
First, BMSCs were spreaded with a cell density of 5-10 × 10 5 /mL on 24-well plates (500 μL per well) and incubated them with DMEN medium containing 10% FBS to form cell monolayers.Then transfected the cells with 50 nM sh14-3-3η (Ribobio, Guangzhou, China).After 24 h, we used a 200-µL sterile plastic micropipette tip to scratch the cell monolayer drawing wound lines; then washed the cell layer with PBS three times and cultured parallel samples in DMEM with 2% FBS medium.After 0, 12, and 24 h of incubation at 37 °C with 5% CO 2 , Cell migration rates were calculated by photographing the cell monolayer wounds.

Statistical analyses
Statistical analyses were conducted by SPSS 17.0 and R 4.0.2software.All experiments were carried out in triplicates, and the results were represented as means ± SDs.Comparisons between two groups were analyzed by Student's T-tests.One-way analysis of variance or two-way analysis of variance was used for comparisons among multiple groups.Diferences were considered statistically signifcant when P < 0.05.

Ethical approval and consent to participate
All animals were kept in a pathogen-free environment and fed ad lib.The procedures for care and use of animals were approved by the Ethics Committee of the Animal Welfare & Ethics Committee of Anhui Medical University and all applicable institutional and governmental regulations concerning the ethical use of animals were followed.All procedures were conducted in accordance with the "Guiding Principles in the Care and Use of Animals.All procedures were conducted in accordance with the ARRIVE guidelines.

Consent for publication
Informed consent was obtained from all individual participants included in the study.www.nature.com/scientificreports/Results

14-3-3η was downregulated in primary osteoporosis
We analyzed 14-3-3η mRNA expression in patients with primary osteoporosis and health donor RNA sequencing data from the GEO database (GSE35958 dataset).The results revealed that the 14-3-3η mRNA expression level was lower in patients with osteoporosis than in healthy donors (P < 0.05, Fig. 1A).Moreover, IHC staining data from the HPA database demonstrated low levels of 14-3-3η expression in fibroblasts and moderate levels of expression in bone marrow cells (Fig. 1B).These results indicate that the 14-3-3η mRNA and protein levels were lower in samples of patients with osteoporosis than in healthy donor samples.

Scratch assay of 14-3-3η-knockdown BMSCs
After counting the control group and sh14-3-3η group BMSCs, we transferred equivalent numbers of BMSCs to 12-well plates.Once the cells were completely adherent to the flask wall, we detected their migration ability using scratch assays.The migrating sh14-3-3η cells were significantly faster than the control cells (Fig. 4B).These results show that 14-3-3η knockdown promoted BMSC migration.

Expression levels of 14-3-3η, MAPK3, and osteogenic factors upon hypoxia induction
Under hypoxic conditions, our western blot results show that the 14-3-3η and MAPK3 protein expression levels were downregulated, whereas those of Hif-α and BMP-2 were upregulated in BMSCs (P < 0.05), compared with the levels in the control cells (Fig. 5A).
The results of the co-immunoprecipitation experiments show that the binding of 14-3-3η to MAPK3 decreased under hypoxic conditions (Fig. 5B).
To verify the association between the 14-3-3η and MAPK3 under hypoxic conditions, we knocked down 14-3-3η in BMSCs and found that the MAPK3 protein expression was downregulated, whereas that of osteogenic factor BMP-2 was upregulated (Fig. 5C).
Moreover, we treated 14-3-3η knockdown BMSCs with the caspase3 inhibitor AC-DEVD-CHO and found that the MAPK3 protein expression was not downregulated.The MAPK3 protein was not cleaved by caspase3 due to the protection provided by AC-DEVD-CHO (Fig. 5D), and the BMP-2 expression was not upregulated.
Thus, 14-3-3η knockdown resulted in downregulated MAPK3 protein expression and increased osteogenic factors expression.However, when caspase3 inhibitor was added to protect the MAPK3 protein from cleavage by Caspase 3, the MAPK3 protein expression did not decrease and the osteogenesis expression did not either.This suggests that 14-3-3η may act like a caspase 3 inhibitor during the MAPK3 downregulation of osteogenic factors expression.

Expression levels of 14-3-3η and miR-142-3P upon hypoxia induction
Under hypoxic conditions, the protein expression of levels of 14-3-3η and MAPK3 were downregulated, whereas the protein expression levels of osteogenesis markers were upregulated.However, under hypoxic conditions, the miR-142-3P levels were significantly higher (P < 0.05) than those under normal conditions (Fig. 6A).www.nature.com/scientificreports/feedback loop through the 14-3-3η and MAPK pathways to promote osteogenic differentiation of BMSCs and may target the MAPK signaling pathway inhibitor, thereby promoting osteoblast differentiation.On the other hand, miR-142-3P may be involved in long-term chronic inflammation, stimulating local osteogenesis, inducing inflammatory transcription factors, and osteogenesis factors, such as BMP-2 (Supplementary information 1).

Conclusion
Our results demonstrate that miR-142-3P regulates MAPK3, a key MAPK signaling pathway protein, by targeting 14-3-3η to inhibit BMSC osteogenesis.This finding improves our understanding of the role of 14-3-3η in BMSC osteogenesis and may aid the development of diagnostic and therapeutic strategies for osteoporosis.Future studies should evaluate the role of miR-142-3P and 14-3-3η in BMSCs using a large sample size.

Figure 1 .
Figure 1.14-3-3η expression levels.(A) Differential 14-3-3η mRNA expression between patients with osteoporosis and healthy donors in the GSE37745 database.Y-axis: Transcripts per kilobase of exon model per million mapped reads.(B) Immunohistochemistry staining of 14-3-3η protein in fibroblasts and bone marrow cells in the HPA database.

Figure 2 .
Figure 2. Gene enrichment analysis for 14-3-3η.(A) Enriched GO terms and KEGG pathways.(B) Heatmaps showing the correlation between the MAPK gene family and 14-3-3η in the GSE37745 database.The left four columns represent donors and the five right columns represent patients.(C) The predicted target miRNAs are presented in a Venn diagram, with only the intersections selected to increase specificity.